Structural basis for activation of calcineurin by calmodulin

Julie Rumi-Masante; Farai I. Rusinga; Terrence E. Lester; Tori B. Dunlap; Todd D. Williams; A. Keith Dunker; David D. Weis; Trevor P. Creamer

doi:10.1016/j.jmb.2011.11.008

Structural basis for activation of calcineurin by calmodulin

Julie Rumi-Masante, Farai I. Rusinga, Terrence E. Lester, Tori B. Dunlap, Todd D. Williams, A. Keith Dunker, David D. Weis, Trevor P. Creamer

Research output: Contribution to journal › Article › peer-review

78 Scopus citations

Abstract

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼ 25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

Original language	English
Pages (from-to)	307-317
Number of pages	11
Journal	Journal of Molecular Biology
Volume	415
Issue number	2
DOIs	https://doi.org/10.1016/j.jmb.2011.11.008
State	Published - Jan 13 2012

Bibliographical note

Funding Information:
The plasmid pETagHisCN containing the human αCaN A (with N-terminal His 6 tag) and B1 genes, together forming αCaN, was obtained from Addgene (Cambridge, MA). This was transformed into Escherichia coli BL21 (DE3) CodonPlus RIL cells (Agilent Technologies, La Jolla, CA) for expression. αCaN was purified on a Ni-NTA column followed by a CaM-Sepharose column (GE Healthcare, Piscataway, NJ). αCaN mutants were generated using the Stratagene QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies). An E. coli codon-optimized gene for the human sequence RD-AID-CT construct was synthesized by Genscript (Piscataway, NJ). This was cloned into the pET303/CT-His vector that adds a C-terminal His 6 tag (Invitrogen, Carlsbad, CA) for expression in E. coli BL21 (DE3). After expression, the RD-AID-CT was purified on a Ni-NTA column followed by a CaM-Sepharose column. Human CaM was expressed from the pETCaMI vector and purified on a 2-trifluoromethyl-10-aminopropyl phenothiazine-Sepharose column. 35 The 2-trifluoromethyl-10-aminopropyl phenothiazine-Sepharose was synthesized at the Center for Structural Biology Chemistry Core Facility, University of Kentucky (supported in part by funds from National Institutes of Health National Center for Research Resources grant P20 RR020171 ). All expressed proteins had their identities confirmed via mass spectrometry. Protein concentrations were determined using the bicinchoninic acid assay. 36

Funding Information:
The authors would like to thank Professor Anthony Persechini of the University of Missouri at Kansas City for providing the pETCaMI expression vector. This work was supported by grants to T.P.C. from the National Science Foundation ( MCB-0843551 ) and the Kentucky Science and Engineering Foundation ( KSEF-148-502-08-227 ) and to F.I.R. from the University of Kansas Graduate Research Fund and in part by funds from National Institutes of Health National Center for Research Resources grant P20 RR020171 (principal investigator: Professor Louis B. Hersh).

Keywords

folding
intrinsically disordered protein
phosphatase

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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10.1016/j.jmb.2011.11.008

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@article{d5e1e8d706b34bf5bb3c418d208545b2,

title = "Structural basis for activation of calcineurin by calmodulin",

abstract = "The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼ 25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.",

keywords = "folding, intrinsically disordered protein, phosphatase",

author = "Julie Rumi-Masante and Rusinga, {Farai I.} and Lester, {Terrence E.} and Dunlap, {Tori B.} and Williams, {Todd D.} and Dunker, {A. Keith} and Weis, {David D.} and Creamer, {Trevor P.}",

note = "Funding Information: The plasmid pETagHisCN containing the human αCaN A (with N-terminal His 6 tag) and B1 genes, together forming αCaN, was obtained from Addgene (Cambridge, MA). This was transformed into Escherichia coli BL21 (DE3) CodonPlus RIL cells (Agilent Technologies, La Jolla, CA) for expression. αCaN was purified on a Ni-NTA column followed by a CaM-Sepharose column (GE Healthcare, Piscataway, NJ). αCaN mutants were generated using the Stratagene QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies). An E. coli codon-optimized gene for the human sequence RD-AID-CT construct was synthesized by Genscript (Piscataway, NJ). This was cloned into the pET303/CT-His vector that adds a C-terminal His 6 tag (Invitrogen, Carlsbad, CA) for expression in E. coli BL21 (DE3). After expression, the RD-AID-CT was purified on a Ni-NTA column followed by a CaM-Sepharose column. Human CaM was expressed from the pETCaMI vector and purified on a 2-trifluoromethyl-10-aminopropyl phenothiazine-Sepharose column. 35 The 2-trifluoromethyl-10-aminopropyl phenothiazine-Sepharose was synthesized at the Center for Structural Biology Chemistry Core Facility, University of Kentucky (supported in part by funds from National Institutes of Health National Center for Research Resources grant P20 RR020171 ). All expressed proteins had their identities confirmed via mass spectrometry. Protein concentrations were determined using the bicinchoninic acid assay. 36 Funding Information: The authors would like to thank Professor Anthony Persechini of the University of Missouri at Kansas City for providing the pETCaMI expression vector. This work was supported by grants to T.P.C. from the National Science Foundation ( MCB-0843551 ) and the Kentucky Science and Engineering Foundation ( KSEF-148-502-08-227 ) and to F.I.R. from the University of Kansas Graduate Research Fund and in part by funds from National Institutes of Health National Center for Research Resources grant P20 RR020171 (principal investigator: Professor Louis B. Hersh).",

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T1 - Structural basis for activation of calcineurin by calmodulin

AU - Rumi-Masante, Julie

AU - Rusinga, Farai I.

AU - Lester, Terrence E.

AU - Dunlap, Tori B.

AU - Williams, Todd D.

AU - Dunker, A. Keith

AU - Weis, David D.

AU - Creamer, Trevor P.

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N2 - The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼ 25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

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Structural basis for activation of calcineurin by calmodulin

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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